(1) Field of the Invention
This invention relates generally to the regulation of cell death and to compounds which regulate cell death, and more particularly, to a novel BH3-containing fragment of BID with cell death agonist activity.
(2) Description of the Related Art
Programmed cell death, or apoptosis, is critical for the successful crafting of multiple lineages, the maintenance of normal tissue homeostasis and a non-inflammatory response to toxic stimuli (Thompson, Science 167:1456-1462, 1995). A distinct genetic pathway apparently shared by all multicellular organisms governs apoptosis. The BCL-2 family of proteins constitutes a decisional checkpoint within the common portion of this pathway. Full members of the BCL-2 family share homology in four conserved domains designated BHI, BH2, BH3 and BH4 (Farrow and Brown, Curr. Opin. Genet. Dev. 6:45-49, 1996). The multi-dimensional NMR and X-ray crystallographic structure of a BCL-X.sub.L monomer indicated that BH1, BH2 and BH3 domains represent .alpha. helices in close proximity which create a hydrophobic pocket presumably involved in interactions with other BCL-2 family members (Muchmore et al., Nature 381:335-341, 1996).
The BCL-2 family possesses pro-apoptotic (BAX, BAK, BOK) as well as anti-apoptotic (BCL-2, BCL-X.sub.L, BCL-W, MCL-1, A1) molecules (Farrow and Brown, supra). The ratio of anti- to pro-apoptotic molecules such as BCL-2/BAX determines the response to a death signal (Oltvai et al., Cell 74:609-619, 1993). A striking characteristic of many BCL-2 family members is their propensity to form homo- and heterodimers (Sedlak et al., Proc. Natl. Acad. Sci. USA 92:7834-7838, 1995; Zha et al., J. Biol. Chem. 271, 7440-7444, 1996). The NMR analysis of a BCL-X.sub.L /BAK BH3 peptide complex revealed both hydrophobic and electrostatic interactions between the BCL-X.sub.L pocket and a BH3 amphipathic a-helical peptide from BAK (Sattler et al., Science 275:983-986, 1997). Results of deletion analysis within BAK (Chittenden et al., EMBO J. 14:5589-5596, 1995) as well as an extensive mutational analysis of BAX (Wang et al., Mol. Cell. Biol., 1998, in press) indicate that the BH3 domain serves as a minimal "death domain" critical for both dimerization and killing.
A divergent subset of the BCL-2 family possesses sequence homology only to the BH3 amphipathic .alpha. helical domain. These "BH3 only" members include the mammalian proteins BID, BAD, BIK, RIM, BLK and HRK, as well as the EGL-1 protein of C. elegans. Of note, all of these molecules are pro-apoptotic, lending credence to the thesis that BH3 represents a minimal death domain (Wang et al., Genes & Dev. 10:2859-2869, 1996; Yang et al., Cell 80:285-291, 1995; Boyd et al., Oncogene 11 :1921-1928, 1995; O'Connor et al., EMBO J. 17:384-395, 1998; Hegde et al., J. Biol. Chem. 273:7783-7786, 1998; Inohara et al., EMBO J. 16:1686-1694 1997; Conradt and Horvitz, Cell 93:519-529, 1998). Where examined, these "BH3 only" molecules are capable of heterodimerizing with classic BCL-2 family members. Mutagenesis of the BH3 domain of BID (Wang et al., 1996, supra; copending U.S. application Ser. No. 08/706,741) and BAD (Zha et al., J. Biol. Chem. 272:24101-24104, 1997) indicated that BH3 was essential for these interactions as well as the killing activity.
BID and BAD lack the typical hydrophobic C-terminal sequence that is found in most BCL-2 family members, which for BCL-2 has been shown to function as a signal anchor segment required for its targeting mitochondria (Nguyen et al., J. Biol. Chem. 268:25265-25268, 1993). Consistent with their lack of a putative C-terminal anchor segment, BID and BAD have been observed in cytosolic as well as membrane based localizations (Wang et al., 1996, supra; Zha et al., Cell 87:619-628, 1996). It has been suggested that BID and BAD may represent death ligands, sensors that receive death signals in the cytosol and translocate to membranes where they interact with membrane bound, classic BCL-2 members which serve as "receptors" (Wang et al., 1996, supra).
This model was supported by the demonstration that in the presence of the survival factor IL-3, cells inactivate BAD by phosphorylation (Zha et al., 1996, supra). Its phosphorylation status has the dual impact of dictating BAD's location as well as its binding partners. Phosphorylated BAD is sequestered in the cytosol bound to 14-3-3; whereas, only the active non-phosphorylated BAD heterodimerizes with BCL-X.sub.L or BCL-2 at membrane sites to promote cell death (Zha et al., 1996, supra).
Recently, pro-apoptotic BAX, despite possessing a hydrophobic C-terminus, has been observed in the soluble fraction of cells as well as mitochondrial membranes (Wolter et al., J. Cell Biol. 139:1281-1292, 1997; Gross et al., EMBO J. 17:3878-3885, 1998). Induced BAX expression (Xiang et al., 1996) or the enforced dimerization of BAX (Gross et al., supra) results in a downstream program of mitochondrial dysfunction as well as caspase activation. A physiologic death stimulus, the withdrawal of IL-3, results in the translocation of monomeric BAX from the cytosol to the mitochondria where it is present as a homodimerized, integral membrane protein (Gross et al., supra).
The importance of the BH3 domain and caspase activation in apoptosis is also suggested by the recent demonstration that BAX and BID fragments containing the B3 domain, i.e., BAX 53-104 and BID 74-128, had death agonist activity when expressed in cells and that death of these cells was significantly inhibited in the presence of the general caspase inhibitor z-VAD-fmk (copending application, U.S. Ser. No. 08/946,039).
The best characterized signal transduction pathways that mediate apoptosis are the cell surface receptors of the TNF family, including CD95 (Fas/Apo-1) and CD120a (p55 TNF receptor) (Tartaglia et al., Cell 74:845-853, 1993; Nagata, Curr. Biol. 6:1241-1243, 1996; Wallach et al., Curr. Opin. Immunol. 10:279-288, 1998). Engagement of Fas/TNF receptor leads to formation of a protein complex known as the DISC (Death-Inducing Signaling Complex) (Medema et al., EMBO J. 16:2794-2804, 1997; Boldin et al., Cell 85:803-815, 1996; Muzio et al., Cell 85:817-827, 1996). This complex comprises Fas/TNF receptor, FADD (MORTI), and pro-caspase-8 (MACH/FLICE/Mch5). Once caspase-8 is recruited, it is processed and released from the complex in active form to activate downstream "effector" caspases (Medema et al., supra; Srinivasula et al., Proc. Natl. Acad. Sci. USA 93:14486-14491, 1996; Muzio et al., J. Biol. Chem. 272:2952-2956, 1997).
The caspase family has been divided into three groups based upon sequence homology and substrate specificity using a positional scanning substrate combinatorial library (Thomberry et al., J. Biol. Chem. 272:17907-17911, 1997). The specificity of caspases 2, 3 and 7 for DEXD (SEQ ID NO:6), where X can be any amino acid, suggests they function at the effector phase of apoptosis. In contrast, the optimal cleavage sequence for caspases 6, 8, and 9 of (I/L/V)EXD resembles activation sites in the effector caspase proenzymes, suggesting that caspases 6, 8, and 9 represent "initiator" caspases.
Wang and colleagues described a cell free system of apoptosis, in which S100 extracts of untreated HeLa cells induced the activation of caspase-3 and DNA fragmentation upon addition of dATP (Liu et al., Cell 86:147-157, 1996). Further purification of the cytosol identified cytochrome c, which was released from the mitochondria during hypotonic lysis of the cells. Apaf-1, a mammalian homolog of CED-4, was a second factor isolated and required for caspase activation (Zou et al., Cell 90:405-413, 1997). Recently, it has been demonstrated that cytochrome c, Apaf-1 and caspase-9 form a complex that initiates a downstream apoptotic caspase cascade (Li et al., Cell 91:479-489, 1997). In addition, it was observed that when Xenopus egg cytosol was incubated with isolated mitochondria, cytochrome c was released, leading to the activation of caspases and nuclear apoptosis (Kluck et al., EMBO J. 16:4639-4649, 1997). The phenomena of cytochrome c redistribution from mitochondria to cytosol was also reported to occur in intact cells during apoptosis (Bossy-Wetzel et al., EMBO J.: 17:37-49, 1998). However, until the studies reported herein, the molecular mechanism responsible for the release of cytochrome c from mitochondria to the cytosol during apoptosis was not known.
Some disease conditions are believed to be related to the development of a defective down-regulation of apoptosis in the affected cells. For example, neoplasias may result, at least in part, from a apoptosis-resistant state in which cell proliferation signals inappropriately exceed cell death signal. Furthermore, some DNA viruses such as Epstein-Barr virus, African swine fever virus and adenovirus, parasitze the host cellular machinery to drive their own replication and at the same time block apoptosis to repress cell death and allow the target cell to reproduce the virus. Moreover, certain disease conditions such as lymphoproliferative conditions, cancer including drug resistant cancer, arthritis, inflammation, autoimmune diseases and the like may result from a down regulation of cell death regulation. In such disease conditions it would be desirable to promote apoptotic mechanisms and one advantageous approach might involve treatment with a cell death agonist having a BH3 domain which has been identified as being critical for killing.
Conversely, in certain disease conditions it would be desirable to inhibit apoptosis such as in the treatment of immunodeficiency diseases, including AIDS, senescence, neurodegenerative disease, ischemic and reperfusion cell death, infertility, wound-healing, and the like. In the treatment of such diseases it would be desirable to diminish the cell death agonist activity of endogenous proteins containing BH3 domains.
Thus it would be desirable to further elucidate how BCL-2 family members, particularly BH3 only family members, regulate apoptosis and to use this knowledge as a basis for treatment modalities to advantageously modulate the apoptotic process in disease conditions involving either inappropriate repression or inappropriate enhancement of cell death.